Muscle stem cells (MuSCs) contribute extensively to muscle tissue regeneration following injury and transplantation. Consequently, MuSCs offer much promise for treating skeletal muscle diseases, including genetic defects and muscle wasting conditions such as sarcopenia and cachexia. The therapeutic utility of MuSCs is limited by the rarity of these cells in adult tissues and the inability to propagate them in culture to therapeutic numbers without loss of their stem cell properties. MuSCs readily give rise in culture to abundant myogenic progenitors called myoblasts, but these cells have extremely limited regenerative potential upon transplantation, as evidenced by failures in myoblast-based clinical trials for the treatment of Duchenne Muscular Dystrophy (DMD). The inability to generate therapeutic levels of MuSCs is due, in part, to a lack of understanding of the molecular mechanisms that regulate the maintenance or induction of the MuSC phenotype. Since MuSC isolation and characterization methods have only been fully validated within the last few years, this paucity of knowledge is not surprising. Here we propose to generate MuSCs from more abundant somatic cells by nuclear reprogramming. In Aim 1, two abundant and culture- expandable human somatic cell types present in muscle tissue, myoblasts and pre-adipocytes, will be reprogrammed to a muscle stem cell phenotype by cell fusion with mouse MuSCs to form non-dividing bi- species heterokaryons. Heterokaryons will allow elucidation of the earliest steps in reprogramming to a MuSC fate and identification of the mechanisms regulating the reversion (myoblast-to-MuSC) or conversion (pre-adipocyte-to-MuSC) of a phenotype. In Aim 2, genetic manipulations of critical reprogramming genes identified in heterokaryons and microarrays of MuSCs (encoding transcription factors and epigenetic regulators) will be tested for their potential to direct reprogramming of myoblasts and pre-adipocytes to functional MuSCs. Reprogrammed phenotypes will be assessed for in vitro MuSC gene expression and epigenetic profiles, and, ultimately, in vivo function following transplantation. In Aim 3, myoblast-to-MuSC and pre-adipocyte-to-MuSC reprogramming will be compared for cells isolated from young and old mice to provide greater understanding of the effects of clinically relevant parameters of age. The proposed studies to investigate maintenance and generation of MuSCs using myoblasts and pre-adipocytes benefit from several recent advances in our laboratory: (a) development of novel techniques to assess global transcriptional and epigenetic changes essential to distinguishing nuclear reprogramming contributions of each cell type in bi-species heterokaryons, (b) a hydrogel-based cell culture substrate that maintains muscle stem cells in vitro, and (c) a noninvasive imaging assay of in vivo MuSC function following transplantation. The molecular insights gained will increase the clinical utility of muscle stem cells and increase our understanding of muscle biology and aging. PUBLIC HEALTH RELEVANCE: A rare but highly functional population of specialized cells, muscle stem cells, is essential to effective skeletal muscle regeneration. Transplantation of muscle stem cells has the potential to treat numerous muscle diseases but is currently impractical due to the limited quantities of these cells that can be isolated from human muscle tissue. We propose to generate clinically sufficient quantities of muscle stem cells from more abundant mature muscle and fat cells through nuclear and cellular reprogramming involving the manipulation of expression of genes associated with muscle stem cell generation.